Antigenic stimulation with cytochrome P450 2J expressed in mouse hepatocellular carcinoma cells regulates host anti-tumour immunity.

Cytochrome P450 2J subfamily (CYP2J) enzymes expressed in mouse hepatocellular carcinoma (HCC) cells were identified as an antigen recognized by specific CD4(+) T cells and the structure of its T cell epitope was determined by proteomics-based exploration. The major histocompatibility complex (MHC) class II binding peptides were isolated from I-A(k)/peptide complex of dendritic cells (DCs) loaded or unloaded with MIH-2 mouse HCC cells. MHC class II-binding peptides found in MIH-2-loaded DCs but not in unloaded DCs were determined by tandem mass spectrometric analysis. The peptide, consisting of amino acid 276-290 (DFIDAFLKEMTKYPE) of mouse CYP2J enzymes, was identified as an antigenic peptide presented in the context of MHC class II. Preventive treatment of mice with CYP2J peptide stimulated interferon (IFN)-gamma production of splenocytes and suppressed the growth of implanted CYP2J-positive MIH-2 cells but not CYP2J-negative murine bladder tumour cells. However, continuous treatment of MIH-2-bearing mice with CYP2J peptide significantly suppressed IFN-gamma production of splenocytes and accelerated the growth of implanted MIH-2 tumours in vivo. Increased frequencies of CD4(+)forkhead box P3 regulatory T cells and CD11b(+)Gr-1(+) myeloid suppressor cells were observed in splenocytes from the continuously immunized mice. These results indicate that antigenecity of CYP2J isoforms expressed in HCC cells activate host anti-tumour immunity at an initial stage of HCC, but suppress host anti-tumour immunity with excessive antigenic stimulation at an advanced stage.

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Correction to: Leukemia (2000); 14: 1260­1265; doi: 10.1038/sj.leu.2401828. Since the publication of the above article the authors have identified an error in Figure 1. Figure 1 shows the modulation of telomerase activity by herbimycin A in K562 cells: (a) cell cycle and (b) telomerase activity, mRNA expressions of hTERT, hTERC, TEP-1, c-myc, cyclin D1 and b-actin, and c-Myc protein. The authors however wish to inform the readers that Figure 1b incorrectly shows hTERT mRNA, which is the result of herbimycin A treatment of cyclin-D1-transfected K562 cells (Figure 3b, hTERT mRNA). While preparing Figure 1, the authors mistakenly submitted a figure that used the incorrect photo data following confusion regarding file names. The correct figure can be found below: The authors wish to apologise for any inconvenience caused and confirm that the conclusions drawn from this research are not affected by this error.

Defective binding of IRFs to the initiator element of interleukin-1beta-converting enzyme (ICE) promoter in an interferon-resistant Daudi subline.

To investigate mechanisms of interferon (IFN) resistance, we have established an IFN-resistant Daudi subline (Daudi(res)), which is 1 X 10(4) times more resistant to IFN-alpha than parental cells. Among the IFN-inducible genes examined, only ICE mRNA expression was deficient in Daudi(res) cells. We then analyzed the regulatory mechanisms of ICE transcription, and found that IFN-induced activation of the ICE promoter was dependent on the binding of IRFs to its initiator (Inr) element. Inr binding of IRFs was markedly diminished in Daudi(res) cells, and forced expression of IRF-1 was able to activate the ICE promoter to the level of parental cells. These results suggest that IRFs and their target genes, as represented by ICE in this study, are involved in IFN resistance.

Cytostatic concentrations of anticancer agents do not affect telomerase activity of leukaemic cells in vitro.

Telomerase, the enzyme that maintains the ends of linear eukaryotic chromosomes, is more active in the majority of malignant tumours than in normal somatic cells. Telomerase plays a key role in the maintenance of chromosomal stability in tumours, but it still remains unknown whether anticancer agents can inhibit telomerase activity. In this study, we evaluated the effect of various anticancer agents (etoposide, cisplatin, irinotecan, mitomycin C and daunorubicin) on the telomerase activity of three human haematopoietic cancer cell lines (Daudi, K562 and U937). A decrease of telomerase activity was not observed in cells treated with IC50 doses of the drugs, except for irinotecan-treated Daudi cells and daunorubicin- and irinotecan-treated U937 cells. Propidium iodide staining disclosed that the cells with decreased telomerase activity were severely damaged. U937 cells exposed to 5 microM (IC90) etoposide showed three different stages of cell viability during treatment. Apoptotic cells with an intact plasma membrane still maintained high telomerase activity, while cells with plasma membrane damage lost telomerase activity. The mRNA of the telomerase catalytic subunit (hTERT) showed a decrease in expression along with the decline of telomerase activity. These results indicate that the concentrations of drugs resulting in cytostatic effects on cells do not affect telomerase activity.

Interferon-alpha repressed telomerase along with G1-accumulation of Daudi cells.

The implications of telomerase on senescence and human carcinogenesis are widely accepted, but the changes of telomerase activity along with cell cycle modulation by anticancer treatment still remain obscure. In this paper, we issued whether the telomerase activity fluctuated along with cell cycle of cultured cancer cells using the antiproliferative effect of interferon-alpha (IFN-alpha). Daudi Burkitt lymphoma cells, treated with IFN-alpha, showed proliferation inhibition and cell cycle arrest at G1. The telomerase activity at 72 h was repressed to about 20% of control cells. Furthermore, after 72 h IFN-alpha treatment, the cells in G1 phase showed the marked decrease of telomerase activity, while cells in S and G2/M still possessed it. Among expressions of telomerase-related genes, only the catalytic subunit of telomerase (hTERT) decreased from 48 h, while the template RNA component (hTERC) and telomerase-associated protein 1 (TEP-1) were not affected. The downregulation of c-Myc preceded the change of hTERT. Moreover, the analysis of cells treated with IFN-alpha for 24 h revealed that cells in G1-to-S transition mainly expressed high hTERT, while S and G2/M cells had higher level of telomerase activity than that of G1 cells. These results indicate that (i) the expression of hTERT precedes the telomerase activity which is higher in S and G2/M phases than G1 phase, (ii) IFN-alpha repressed the telomerase activity in a cell cycle-dependent manner with the downregulation of hTERT.

Hemolysis caused by CMV infection in a pregnant woman with silent elliptocytosis.

Elliptocytosis is reported to occur in at least 1 per 5000 individuals, but most cases are heterozygous and do not show clinical hemolysis. Healthy individuals with silent elliptocytosis, however, may suddenly have an episode of hemolysis [1]. Here we report a woman in the third trimester of pregnancy who suffered from cytomegalovirus (CMV) infection with hemolysis. Scanning electron microscopy showed that half of her red blood cells were oval, and protein analysis revealed a 50% reduction of protein 4.1. We discuss the role of CMV infection and pregnancy in the onset of hemolysis in a patient with otherwise silent elliptocytosis.

Herbimycin A down-regulates messages of cyclin D1 and c-myc during erythroid differentiation of K562 cells.

The ansamycin antibiotic, herbimycin A, is a potent tyrosine kinase inhibitor, and induces the erythroid differentiation of bcr-abl-possessing K562 cells. The growth of K562 cells was cytostatically reduced to less than 50% of the control level at 48 h by 0.5 microgram/ml of herbimycin A treatment. A total of 12% and 53% of the treated cells were benzidine-positive at 24 h and 48 h, respectively. The percentage of cells in the S phase decreased rapidly from 60% to 15% after 12 h of treatment. The reduction of S phase cells persisted until 24 h, whereas the G1 population conversely increased. Then underphosphorylated retinoblastoma gene product increased from 6 h to 24 h, but returned to baseline at 48 h. Most cell cycle controlling genes were unchanged by herbimycin A treatment. However, both cyclin D1 and c-myc were prominently down-regulated in the early phase of treatment, corresponding to the decline of the S phase population. Cyclin D1 was initially down-regulated to an undetectable level at 6 h, although its expression recovered gradually from 12 h and returned to baseline at 24 h. c-myc was also down-regulated from 1 h to 6 h. These data suggest that signals originating from bcr-abl kinase are at least partly transduced through both c-myc and cyclin D1, and that herbimycin A-induced erythroid differentiation occurs during or after the cessation of growth due to interference with these signals.

A novel variant of acute myelomonocytic leukemia carrying t(3;12)(q26;p13) with characteristics of 3q21q26 syndrome.

Chromosomal translocation often results in aberrant activation of the genes with oncogenic potential and, thus, plays an important role in leukemogenesis. We report a unique case of acute myelomonocytic leukemia carrying a rare reciprocal translocation, t(3;12)(q26;p13). This patient displayed typical clinical features of 3q21q26 syndrome such as abnormal thrombopoiesis and rapid disease progression. Blastic cells from the patient strongly expressed the EVI1 gene, which is located on 3q26 and is normally suppressed in bone marrow cells. Expression of the TEL gene, located on 12p13, was also observed, but fusion transcript between two genes was not found. No structural alterations of the EVI1 and TEL genes were detected by Southern blot and PCR analyses. We reviewed previous literature and found 10 other cases with t(3;12)(q26;p13). These patients comprise a unique disease group with features including dyshematopoiesis and poor prognosis. However, characteristics related to 3q21q26 syndrome were observed only in the present case. Further investigation is required to elucidate the molecular basis of this particular entity.

Concentration-dependent variable effects of etoposide on the cell cycle of CML cells.

BACKGROUND: Etoposide, a DNA-topoisomerase II inhibitor, is used for a broad spectrum of cancers with various therapeutic strategies. But the molecular mechanisms of its concentration-dependent effects are not clearly defined. MATERIALS AND METHODS: Chronic myelogenous leukemia K562 cells were treated with low (5 microM) or high (100 microM) concentrations of this drug and the changes of cell cycle progression, expression of cell cycle regulating genes and cyclin B1-dependent histone H1 kinase activity were studied. RESULTS: In the presence of 5 microM etoposide, K562 cells continued to synthesize DNA and most cells showed progress into G2 phase until 24 hours. In contrast, 100 microM etoposide rapidly inhibited DNA synthesis by around 6 hours and most cells remained in their initial phase, while the incorporation of bromodeoxyuridine was partially resumed from 12 hours. The histone H1 kinase activity was only down-regulated in the early phase of 100 microM treated cells. Among the cell cycle controlling genes, c-Myc and P21Cip1/WAF1 showed impressive responses to the two etoposide concentrations. At 100 microM, c-Myc protein rapidly vanished at 3 hours, while p21Cip1/WAF1 was inversely induced from 3 hours. These changes were also observed at 5 microM, but they occurred slowly and weakly. CONCLUSION: The present findings indicate that two concentrations of etoposide functioned as an anticancer agent through modulating the genes related in cell cycle progression. Differing responses of c-Myc and p21Cip1/WAF1 at two concentrations may govern the antiproliferative effects of etoposide.

MEK and p38MAPK inhibitors potentiate TNF-alpha induced apoptosis in U937 cells.

BACKGROUND: TNF-alpha is one of the key inflammatory cytokines and it modulates various events through several pathways. U937 myelomonocytic leukemia cells are sensitive to TNF-alpha and about 20% of these cells undergo apoptosis within 6 hours after treatment. Co-treatment of these cells with actinomycin D or cycloheximide enhances TNF-alpha induced apoptosis, suggesting that some TNF-alpha-derived signals can augment apoptosis. We investigated whether mitosis-activating protein kinases (MAPKs) had an influence on TNF-alpha induced apoptosis. MATERIALS AND METHODS: U937 cells were treated by TNF-alpha with or without MEK or p38MAPK inhibitors. Apoptosis was assessed morphologically by fluorescence microscopy and caspase-3 was studied by immunoblotting. Expression of apoptosis-inhibitory proteins was studied by RT-PCR whilst the activation of JNKs was investigated by detecting their phosphorylation. RESULTS: TNF-alpha treatment induced apoptosis in about 23% of the cells, while pretreatment with a MEK inhibitor (PD98059) caused 69% of the cells to undergo apoptosis. The inhibition of p38MAPK by SB203580 scarcely enhanced apoptosis, although another p38MAPK inhibitor (PD169316) induced apoptosis in 37% of the cells. Simultaneous pretreatment of cells with PD98059 and PD169316 resulted in the highest level of TNF-alpha induced apoptosis and 90% of the cells underwent apoptosis after 6 hours. In cells pretreated with PD98059 plus PD169316, caspase-3 was completely cleaved at 6 hours and early induction of c-IAP2/HIAP 1 mRNA was not observed. JNKs showed rapid and extensive phosphorylation in these cells. CONCLUSION: TNF-alpha induced apoptosis was potentiated by the inhibition of either MEK alone, or MEK plus p38MAPK, suggesting that the MAPK pathway may be a promising target for cancer therapy.

Serum stimulation and cell density regulate the proliferation of AsPC-1 cells through control of cyclin E and p27KIP1 expression.

BACKGROUND: Cellular proliferation in normal cells is tightly regulated by environmental conditions. Growth factors stimulate proliferation while cell confluence inhibits it. Human pancreatic cancer AsPC-1 cells were believed to escape from these restrictions because they possessed several mutations which promote cell proliferation. In this study, we focused on the relationships between growth conditions and the proliferation of AsPC-1 cells. MATERIALS AND METHODS: AsPC-1 cells were cultured under several growth conditions and the proliferation of cells was studied by incorporation of 3H-thymidine. The alterations of cell-cycle-related genes were studied by immunoblotting. RESULTS: By four consecutive days in culture, the nucleotide incorporation of AsPC-1 cells was markedly suppressed and the suppression was overcome by medium change or reduction of cell density. The induction of cyclin D1 by serum stimulation was observed, concomitant with the transient activation of extracellular signal-regulated kinases (ERKs). The most prominent changes of cell-cycle-regulating genes following consecutive culture or serum reduction were the down-regulation of cyclin E and the induction of p27KIP1. The down-regulation of cyclin E was more sensitive to cell density, while the induction of p27KIP1 was regulated by both increased cell density and reduction of serum. The down-regulation of p27KIP1 was caused by protein degradation. CONCLUSIONS: The proliferation of AsPC-1 cells was still controlled by cell density and serum stimulation; nevertheless, the cells possessed several oncogenic mutations. These results may provide a rationale for modifying the growth environment for treatment of pancreatic cancers.

Repeated administration of cytokines improve the ex-vivo generation of human dendritic cells.

The in vitro generation of dendritic cells (DCs) enables us to practice antitumor immune therapy. Peripheral monocytes can differentiate to DCs in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) in vitro. To generate a large number of DCs, we compared the number of DCs generated from leukapheresis products to those from conventional blood samplings in healthy volunteers. The induction rates of the DCs were equal for these two blood samplings, and 10(7) DCs were obtainable from one leukapheresis product. In contrast, the number of DCs varied significantly depending on the individual (30-50% in good responders vs. less than 10% in poor responders). DCs appeared as aggregates of indented cells during culture in good responders, while large cells were floating sparsely in poor responders. Repeated administration of GM-CSF and IL-4 improved the yields of DCs and induced proliferation of autologous lymphocytes in poor responders. As a prototype of cell therapy, the generation of DCs will require a titration of differentiation processes, depending on each individual's responsiveness to cytokines.

[Transformation into chronic myelomonocytic leukemia in a patient with primary myelofibrosis associated with severe hypoplasia: report of an autopsy case].

A 70-year-old male was admitted because of anemia in September 1989, and primary myelofibrosis was diagnosed based on the presence of leukoerythroblastosis, a normal chromosomal analysis and pathological findings of fibrosis in bone marrow. Although he was anemic, he did not require any treatment for two years. Then his hematological status deteriorated to severe pancytopenia, and the marrow biopsy revealed marked hypoplasia with fatty replacement and scattered fibrosis. He was treated with metenolon without success and frequent transfusion of packed red cell was required. This hypoplastic status continued for seven months. In May 1992 his WBC count increased gradually with monocytosis. The marrow was filled with various stages of monocytes, with almost no fibrosis remaining. The chromosomal analysis was repeated but disclosed no abnormalities, consistent with the negative result of BCR-ABL rearrangement investigated by the RT-PCR method. One month later, when the patient died of multiple cerebral bleeding and infection, the leukocyte count exceed 90,000/microliters. It is known that major causes of death for patients with primary myelofibrosis are infection, bleeding, cardiac trouble and transformation to leukemia. We describe a case of myelofibrosis who developed to chronic myelomonocytic leukemia following severe aplastic phase.

Differing responses of G2-related genes during differentiation of HL60 cells induced by TPA or DMSO.

Differentiation leads to the cessation of cellular proliferation, but little is known about the molecular mechanisms of growth arrest. We compared the effect of two differentiation inducers, 12-o-tetradecanoyl 13-acetate (TPA) and dimethyl sulfoxide (DMSO) on both the cell-cycle and the modulation of G2-related genes in synchronized HL60 cells. TPA treatment of HL60 cells resulted in G1 arrest within 24 h. In contrast, the cell cycling of DMSO-treated cells was initially accelerated and they progressed to the second cycle before accumulating in the G1 phase. Expression of cyclin B, cdc25, wee1 and cdc2 was studied during cell cycle arrest by Northern blot hybridization. Expression of cyclin B, cdc25 and cdc2 fluctuated in association with cell cycle progression towards the G2/M phase, while wee1 expression remained constant in untreated cells. These four genes were highly expressed in TPA-treated cells for the first 12 h, but drastic down-regulation was seen at 18 h and expression became undetectable after 24 h. In contrast, no remarked changes of gene expression were seen in DMSO-treated cells. These findings suggest that cell cycle progression along with the initial process of differentiation in response to TPA differs from the response to DMSO and that the down-regulation of cdc2 expression by TPA-treated HL60 cells contributes to endorsement of G1 arrest.

Differential responses of Bcl-2 family genes to etoposide in chronic myeloid leukemia K562 cells.

Etoposide is a potent anticancer agent that is used to treat various tumors. We have investigated the dose-dependent effect of etoposide on apoptosis using chronic myeloid leukemia K562 cells treated with low (5 microM) or high (100 microM) concentrations of the drug. At a low concentration, etoposide induced little apoptosis at 24 h, while about 20% of the cells showed apoptosis morphologically at a high concentration. Processing of caspase-3 was slightly detected from 12 h and became obvious at 24 h with 100 microM etoposide. Caspase-3-like protease activity was detected at 24 h with a high concentration. Moreover, these changes were accompanied by cleavage of poly ADP ribose polymerase (PARP). Changes of the mRNA levels of most apoptosis-regulating genes were not prominent at both concentrations, except for the rapid induction of c-IAP-2/HIAP-1 and the down-regulation of Bcl-X(L) by 100 microM etoposide. The downregulation of Bcl-X(L) protein occurred from 6 h, while Bax protein conversely showed a slight increase from 6 h. Taken together, the present findings show that the dose-dependent apoptotic effect of etoposide is based on a change in the balance between Bcl-X(L) and Bax, which precedes the activation of caspase-3.

Changes of G1 cyclins, cdk2, and cyclin A during the differentiation of HL60 cells induced by TPA.

Differentiation induction by 12-o-tetradecanoyl 13-acetate (TPA) results in the growth arrest of HL60 cells in the G1 phase. However, little is known about the changes of cell cycle-regulating genes during this differentiation process. We investigated the changes of mRNA for various cyclins (A, C, D1, D2, D3 and E) and cdk2. Synchronized HL60 cells began to proliferate immediately after release from cell cycle block and cell cycle synchrony was obvious until the second S phase. TPA-treated cells accumulated in G1 phase within 24 h and most of the cells were arrested in this phase at 36 h. The expression of cyclins and cdk2 was studied by Northern blot hybridization of the reverse-transcription polymerase chain reaction (RT-PCR). TPA treatment altered the expression of all genes studied. The expression of cdk2 and cyclin A mRNA was markedly down-regulated. Cyclin E mRNA expression was also prominently down-regulated from 12 h to 36 h, at which time a second increase of its expression was observed in control cells. In contrast, the expression of cyclin D1 mRNA was induced by TPA, while its expression in control cells was undetectable by Northern blot hybridization throughout the cell cycle. Cyclin C expression was faint and fluctuated irrelevant of cell cycle, but its expression in both control and TPA-treated cells was higher than at baseline. Cyclin D2 expression remained stable in control cells and TPA treatment resulted in slight down-regulation at 12 h, but no difference was observed after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes of cell cycle-regulating genes in interferon-treated Daudi cells.

Interferon (IFN) modulates the expression of several genes and some of them are considered to be responsible for the inhibition of cellular growth. However, the alterations of cell cycle-regulating genes produced by IFN still remain unclear. Accordingly, we studied the expression of cell cycle-regulating genes during IFN-induced growth arrest. Cell cycle synchronized and unsynchronized Daudi Burkitt lymphoma cells were treated with IFN. Both the cell cycle distribution and the expression of cell cycle-regulating genes (cdk2, cdc2, cyclins A, B, C, D3, cdc25, and wee 1) were studied by flow cytometry and by Northern blot hybridization or the reverse-transcription polymerase chain reaction, respectively. Treated cells passed through the first G1 phase and gradually accumulated in the following G1 phase. Expression of cyclins A, B, and D3 oscillated along with the cell cycle progression in control cells, and the alterations of cyclin B expression were especially prominent. Both cdc2 and cdk2 also showed changes, but these were not so distinct as observed with cyclin B. Expression of cdc25 and wee1 was little affected by cell cycle progression. In IFN-treated cells, expression of cyclins A and B were down-regulated, while that of cyclin C was not. Cyclin D3 expression was also down-regulated at 48 h, followed by an increase at 72 h. Expression of both cdc2 and cdk2 was down-regulated, especially that of the later. Wee1 expression was down-regulated by IFN but, the expression of cdc25 remained stable. These findings suggest that the modulation of cell cycle-regulating genes, particular by cyclin A and cdk2, plays an important role in IFN-induced cellular growth arrest.

A macrolide antibiotic, roxithromycin, inhibits the growth of human myeloid leukemia HL60 cells by producing multinucleate cells.

The antiproliferative effect of roxithromycin (RXM) was studied using human myeloid leukemia HL60 cells. RXM inhibited the growth of HL60 cells in a concentration-dependent manner, and significantly inhibited growth at concentrations above 75 microM. This growth inhibition was not associated with specific cell cycle arrest and DNA synthesis was not impaired. In addition, the number of viable cells remained almost unchanged in the presence of 100 microM RXM. RXM induced growth inhibition at least partly by the formation of multinucleate cells. Both flowcytometric and morphological examination revealed that more than 40% of the RXM-treated cells were binucleate. These findings demonstrate that RXM is a potent new modulator of cell cycle progression in HL60 cells and suggest that the inhibition of cytokinesis by this drug may provide a new model for studying mitosis.

Interferon modulates the messenger RNA of G1-controlling genes to suppress the G1-to-S transition in Daudi cells.

Interferon (IFN) is one of the potent antiproliferative cytokines and is used to treat some selected cancers. IFN arrests the growth of Burkitt Lymphoma derived cell line Daudi cells in the G1 phase. G1-to-S progression is controlled by positive and negative regulatory genes. Therefore, we investigated the effects of IFN on G1-controlling genes. Expression of cyclin-dependent kinases (Cdks 2, 3, 4, 5, 6), MO15/Cdk7, and cyclins E and H was studied to assess positive regulators, while p15Ink4B, p16Ink4, p18, p21Cip1, and p27Kip1 were assessed as negative regulators. Cdks 2, 4, 6 and cyclin E were markedly down-regulated. MO15/Cdk7 expression showed little change, but its regulatory subunit (cyclin H) was down-regulated like cyclin E. Expression of p15Ink4B and p16Ink4 was not observed. p18 was induced until 48 h and its expression returned to the initial level at 72 h. In contrast, p21Cip1 mRNA expression remained at the baseline level throughout IFN treatment, while the expression of p27Kip1 increased at 48 and 72 h. Taken together, these data indicate that IFN changes the messenger RNA of G1-controlling genes towards the suppression of G1-to-S transition.